T H E J O U R N A L O F C E L L B I O L O G Y
© 2008 Chen et al.
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 181 No. 7 1129–1139
Correspondence to Kai Chen: email@example.com
Abbreviations used in this paper: EGFR, EGF receptor; ERK, extracellular signal-
regulated kinase; HAEC, human aortic endothelial cell; MEF, mouse embryonic
fi broblast; MSP, mitochondrial signal peptide; NO, nitric oxide; NOS, NO syn-
thase; Nox, NADPH oxidase; PTP, protein tyrosine phosphatase; ROS, reactive
oxygen species; VE, vascular endothelial.
There is now considerable evidence indicating that reactive
oxygen species (ROS) are involved in signal transduction. These
species include the three successive reduction products of molec-
ular oxygen such as superoxide ( • O 2 ? ), hydrogen peroxide
(H 2 O 2 ), and hydroxyl radical ( • OH). Each of these species pos-
sesses chemical properties that potentially impact their signal-
ing function. For example, OH • is the most unstable ROS with
a half-life of 10 ? 9 s ( Pryor, 1986 ), indicating that it will react
with any species within a radius of ? 30 Å , thereby limiting
its ability to transmit signals across any signifi cant distance.
Superoxide carries a negative charge that limits its membrane
permeability to anion channels. In contrast, H 2 O 2 is thought to
be freely permeable and, thus, could react with several intra-
and extracellular targets, limiting its specifi city; however, this
concept has recently been challenged ( Branco et al., 2004 ).
Thus, the chemical properties of ROS suggest signifi cant limi-
tations toward the production of specifi c cellular responses.
The prototypical NADPH oxidase (Nox) family member
is Nox2 (also known as gp91 phox ), which was initially found in
phagocytes and plays a role in host defense by giving an outward
burst of ROS ( Cheng et al., 2001 ). Over the last several years,
Nox2 along with its homologues, including Nox1, Nox3, Nox4,
Nox5, Duox1, and Duox2, have been identifi ed in nonphago-
cytes. It now appears that Noxs in nonphagocytes serve as a major
source of intracellular ROS that play important signaling roles.
However, the complexity of these isoforms in controlling ROS
production is increasingly apparent because each isoform has its
unique expression pattern, subcellular localization, and subunits
requirement. The specifi c mechanisms for specifi c cell signaling
responses are not known; therefore, the goal of this study is to
examine the mechanisms of ROS signaling specifi city.
Exogenous versus endogenous sources of
ROS initiate distinct signaling responses
ROS are known to mediate a variety of cellular signaling path-
ways, and experiments in vitro typically use an exogenous
source of ROS such as H 2 O 2 to directly initiate signaling re-
sponses. However, one must consider that exogenous applica-
tion of H 2 O 2 may not adequately refl ect authentic endogenous
ROS signaling. Indeed, exogenous H 2 O 2 produces broad signal-
ing responses, including the activation of extracellular signal-
regulated kinase (ERK), JNK, p38 MAPKs ( Fig. 1 A ), and Akt
( Thomas et al., 2002 ). In contrast, the EGF-stimulated signaling
response, which is known to be mediated by ROS in epithelial
cells ( Bae et al., 1997 ), was restricted to the mitogenic ERK path-
way ( Fig. 1 B ) in endothelial cells. We found that EGF-induced
diffusible ROS dictate specifi c cellular responses. In this
study, we demonstrate that nicotinamide adenine dinucle-
otide phosphate reduced oxidase 4 (Nox4), a major Nox
isoform expressed in nonphagocytic cells, including vascu-
lar endothelium, is localized to the endoplasmic reticulum
(ER). ER localization of Nox4 is critical for the regulation
of protein tyrosine phosphatase (PTP) 1B, also an ER resi-
eactive oxygen species (ROS) function as intracellu-
lar signaling molecules in a diverse range of bio-
logical processes. However, it is unclear how freely
dent, through redox-mediated signaling. Nox4-mediated
oxidation and inactivation of PTP1B in the ER serves as a
regulatory switch for epidermal growth factor (EGF) re-
ceptor traffi cking and specifi cally acts to terminate EGF
signaling. Consistent with this notion, PTP1B oxidation
could also be modulated by ER targeting of antioxidant
enzymes but not their untargeted counterparts. These data
indicate that the specifi city of intracellular ROS-mediated
signal transduction may be modulated by the localization
of Nox isoforms within specifi c subcellular compartments.
Regulation of ROS signal transduction by NADPH
oxidase 4 localization
Kai Chen , Michael T. Kirber , Hui Xiao , Yu Yang , and John F. Keaney Jr.
Division of Cardiovascular Medicine, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605
JCB • VOLUME 181 • NUMBER 7 • 2008 1130
lation induced by EGF, whereas Nox2 suppression had no mate-
rial impact ( Fig. 1 F ). Together, these data implicate endothelial
Nox4 as an endogenous source of ROS in mediating EGF-
induced signaling responses.
Nox4 is an ER-residing protein
To gain insight into the nature of Nox4, we examined its sub-
cellular localization. The topography of Nox4 was examined in
silico initially using PredictProtein (Columbia University), and
it possesses six putative membrane-spanning regions, predicting
ERK activation was attenuated by catalase overexpression ( Fig. 1,
C and D ), indicating a role for ROS and a distinction between
endogenous and exogenous ROS signaling.
To examine endogenous ROS responses in the endothe-
lium, we suppressed the expression of Nox4 and Nox2, the two
major Nox isoforms present in endothelial cells ( Sorescu et al.,
2002 ), using RNAi. As shown in Fig. 1 E , both Nox4 and Nox2
siRNA substantially reduced their respective mRNA and protein
levels without cross-reactivity by > 75% and 60%, respectively.
We found that suppression of Nox4 attenuated ERK phosphory-
Figure 1. A different source of ROS initiates distinct signaling responses in endothelial cells. (A) HAECs were treated with 100 μ M H 2 O 2 for the indicated
times, and total cell lysates were subjected to antibodies specifi c for pERK, ERK, pcJun, cJun, pp38, or p38. (B) HAECs were treated with 50 ng/ml EGF for the
indicated times followed by immunoblotting with antibodies as in A. (C and D) Cells were treated with control adenovirus (Ad-LacZ) or catalase-overexpressing
adenovirus (Ad-Cat) for 24 h before treatment with EGF followed by immunoblotting as in A. Error bars represent SD. *, P < 0.05. (E and F) HAECs were trans-
fected with siRNA against nontargeting control (NT), Nox4, or Nox2 for 48 h before extraction of total RNA for RT-PCR and Western blotting (E) or treatment with
EGF for 15 min followed by immunoblotting with pERK, ERK, Nox4, and Nox2 antibodies (F). Results are representative of four independent experiments.
1139NADPH OXIDASE 4 REGULATES REDOX SIGNALING • Chen et al.
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